Research ArticleA novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury
Introduction
The perisinusoidal hepatic stellate cells (HSCs) constitute 8–12% of the liver cell population, express glial fibrillary acidic protein (GFAP) and/or desmin, and are the major storage site of retinoids [1]. During liver injury, HSCs undergo activation characterized by the loss of retinoids, expression of α-smooth muscle actin and differentiation into proliferating myofibroblast-like cells. Activated HSCs produce excessive extracellular matrix (ECM) and exhibit increased expression of tissue inhibitors of metalloproteinases and reduced or unchanged expression of matrix metalloproteinases [2], [3], [4], thus becoming the major cell type responsible for hepatic fibrosis [5]. Activated HSCs are postulated to contribute to portal hypertension by their high contractility and up-regulation of the powerful vasoconstrictor endothelin-1 (ET-1) and its receptors [6], [7].
HSCs express intercellular adhesion molecule-1 [8], produce various cytokines and chemokines [8], [9], [10], and thus can play an important role in hepatic inflammation. Gram-negative bacterial endotoxin (lipopolysaccharide: LPS) stimulates the synthesis of nitric oxide (NO), ET-1, tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both quiescent and activated HSCs; LPS-challenged HSCs stimulate NO synthesis, inhibit DNA synthesis and cause apoptosis of cultured hepatocytes [11], [12], [13], [14].
Recent work demonstrates that HSCs also influence hepatic immunological functions. HSCs induce apoptosis of allogeneic CD4+ and CD8+ T cells [8], [15], present bacterial lipid antigens to NKT cells [16], expand immunosuppressive regulatory T cells [8], and render dendritic cells immunosuppressive [10]. Furthermore, HSCs secrete powerful antioxidant protein(s) that protect hepatocytes from ischemia/reperfusion (I/R) injury [17].
Thus, HSCs are highly versatile cells that can profoundly influence hepatic structure and functions in physiology and pathology. Most of the in vivo work confirming their role in hepatic pathology has been focused on fibrosis. A fungal metabolite gliotoxin was found to cause apoptosis of activated rat and human HSCs in vitro, and of rat HSCs in vivo resulting in resolution of fibrosis [18], [19]. However, gliotoxin also induces apoptosis of KCs and endothelial cells in the fibrotic liver [20], [21]. Ebrahimkhani et al. [22] administered gliotoxin into bile duct-ligated mice in conjugation with the single-chain antibody C1–3, which recognizes synaptophysin expressed by activated HSCs [23]; C1–3-gliotoxin caused resolution of fibrosis by selectively depleting HSCs. It was recently reported, using a similar mouse model described in the present study that concomitant treatment of B6.Cg-Tg(Gfap-Tk)7.1Mvs/J transgenic mice with ganciclovir promoted depletion of HSCs, and caused amelioration of CCl4-induced fibrosis and hepatic injury [24]. However, the role of HSCs in acute injury to the normal liver has not yet been evaluated. Here, we show amelioration of I/R- and endotoxin-induced acute injury to otherwise normal HSC-depleted liver, suggesting HSCs’ critical role in pathologies unrelated to activation-dependence.
Section snippets
Animals
The protocols were approved by the IACUC according to NIH guidelines. Wild-type male C57BL/6 (WT-B6) and B6.Cg-Tg(Gfap-Tk)7.1Mvs/J (GFAP-Tg) mice were from The Jackson laboratory. GFAP-Tg mice express the herpes simplex virus thymidine kinase (HSV-TK) transgene under the GFAP promoter [25]. HSV-TK phosphorylates non-toxic ganciclovir (GCV) to GCV-monophosphate, which is converted to GCV-triphosphate by cellular guanylate kinase; phosphorylated GCV incorporates into the DNA causing death of
Characterization of the CCl4 effect on WT mice
To ensure that at the end of GCV treatment of mice, there is no residual hepatic injury due to earlier CCl4 administration before subjecting them to I/R or endotoxemia, WT-B6 mice were sacrificed a day after the third CCl4 injection or after 10 days of GCV treatment following termination of CCl4. There was significant hepatic injury the day after third CCl4 administration (Fig. 1A and B, middle panels), in both centrilobular and periportal areas (Supplementary Fig. 2), accompanied by
Discussion
The role of activated HSCs in hepatic fibrosis and cirrhosis has been established [5], [33]. Recent research with cell culture studies and some indirect in vivo evidence indicate that quiescent or transiently activated HSCs (found in the liver during early phase of acute injury) can significantly influence metabolic and hemodynamic properties of the liver [7]. However, the lack of animal models in which HSCs are selectively depleted has been a major drawback to demonstrate unequivocally their
Financial support
NIH PO1A1081678 and VA Merit Review Award 1IO1BX001174, and Department of Surgery, University of Cincinnati and Children’s Hospital Medical Center, Cincinnati.
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The underlying research reported in the study was funded by the NIH Institutes of Health.
Acknowledgement
We thank Ms. Rebecca B. Schuster for excellent technical assistance.
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These authors contributed equally to this investigation.